Prof.Mr.Kiran K. Shinde (M.Pharm), Assistant professor (VNIPRC) Pharmaceutical microbiology (Second year b.pharm) (3rd semester) Introduction Types of Spoilage Factors affecting the Microbial spoilage of pharmaceutical products Sources and Types of Contamination Assessment of microbial contamination and spoilage

1. MICROBIAL SPOILAGE
• INTRODUCTION
• TYPES OF SPOILAGE
• FACTORS AFFECTING THE MICROBIAL SPOILAGE OF PHARMACEUTICAL PRODUCTS
• SOURCES AND TYPES OF MICROBIAL CONTAMINATION
• ASSESSMENT OF MICROBIAL CONTAMINATION AND SPOILAGE
PREPARED BY,
PROF. SHINDE KIRAN (M.PHARM)
ASSISTANT PROFESSOR (VNIPRC)
PHARMACEUTICAL MICROBIOLOGY-I
SECOND YEAR B.PHARM
IIIRD SEMESTER - UNIT 5
Vidya Niketan Institute
Of Pharmacy &
Research Centre, Bota

2. INTRODUCTION
 Micro-organisms form an integral part of environment as they are present in air, water,
soil, food, etc.
 Different types of micro-organisms mainly contaminate the pharmaceutical preparations
and spoil it. Such spoilage results in major health problems to the users and
manufactures.
 In injections, eye-drops and certain dressings sterility must be seen otherwise infections
cause harmful reactions.
 Oral and topical preparations may cause infections after microbial contamination.
 Pharmaceutical products may be considered to be microbiologically spoiled if low levels
of pathogenic microbes or toxic microbial metabolites are present and detectable
physical or chemical changes have occurred in product.
 Oral formulation contaminated by micro-organisms mainly cause gastrointestinal
disorders e.g. Salmonella typhi
 External semi-solid preparations applied on skin, provide the opportunity for
Pseudomonas and Staphylococcus and produce localized infections.

3. TYPES OF SPOILAGE
1. Infection induced by contaminated pharmaceutical products
2. Physical and chemical deterioration of products
3. Observable effects of microbial attack on products
4. Ingredients susceptible to microbial attack
• Therapeutic agents
• Surface active agents
• Polymers and humectants
• Fats and oils
• Preservatives and disinfectants
• Sweetening, flavouring and colouring agents

4. 1. Infection induced by contaminated pharmaceutical products
Pharmaceutical products may be contaminated by pathogenic micro-organisms mainly
from raw materials or at the time of preparations. They cause serious infections to the
when they use these drugs. Different dosage forms and contaminated micro-organisms
are responsible for diseases are as follows:
Contaminating micro-organisms and their infections
2. Physical and chemical deterioration of products
The rate of deterioration of ingredients depend upon its chemical structure, physico-
chemical properties of a particular environment. Some naturally occurring ingredients are
particularly sensitive to attack. Crude vegetables & animal extract often contain wide
assortments.

5. 3. Observable effects of microbial attack on products
Microbial spoilage of different dosage forms may be detected by organoleptic tests.
spoiled products may release very unpleasant smelling and tasting such as sour, fatty acids,
earthy, amines, and smells. Formulations become coloured green, pink, browny, black, or
Spoiled creams become gritty or lumpy. Surface activity is reduced.
4. Ingredients susceptible to microbial attack
i. Therapeutic agents: Laboratory experiments have demonstrated that many drugs
are capable of gross degradation by a wide variety of micro-organisms. Many microbial
transformation are useful for production of potent steroids. Aspirin may be converted to
salicylic acid and penicillin to inactive products.
ii. Surface active agents: Alkali-metal and amine soaps of fatty acids re generally
stable due to the slightly alkaline pH of the formulations, which is easily degraded in
sewage. The cationic surfactants used as antiseptics and preservatives in pharmacy are
slowly degraded at high dilution in sewage.

6. iii. Polymers and humectants: Thickening and suspending agents used in pharmacy are
subjected to microbial depolymerisation by extracellular enzymes yielding nutritive
fragments and monomers e.g. starch, pectin, dextran, protein, and carboxymethyl cellulose.
iv. Fats and oils: This are attacked extensively when dispersed in aqueous formulations such
as oil-in-water emulsions. The microbial metabolism of hydrocarbon oils presents a
considerable problem in engineering and fuel technology when water is present as a
contaminant.
v. Preservatives and disinfectant: Most organic preservatives and disinfectants are
metabolised readily by many bacteria and fungi and may serve as growth substrates at
concentrations below their effective ‘use’ levels. Degradation is less commonly reported.
organisms grow in dilute forms.
vi. Sweetening, flavouring and colouring agents: Many sugars and other sweetening
agents used in pharmacy are ready substrates for microbial growth. Some are used in
high concentrations to reduce water activity. Aqueous stock solution support growth of
organisms e.g. peppermint water and chloroform water and colouring agents such as
or tartrazine readily support growth of bacteria and yeasts.

7. FACTORS AFFECTING THE MICROBIAL
SPOILAGE OF PHARMACEUTICAL PRODUCTS
1. Size of inoculum
2. Nutritional factors
3. Moisture content
4. Temperature
5. pH
6. Redox potential
7. Protective components
 The physical and chemical status of a pharmaceutical formulation influences the type
and extent of microbial spoilage considerably. A specific combinations of conditions
within a product may favour its degradation by a particular group of micro-organisms.
 Factors affecting the microbial spoilage are as follows

8. 1. Size of inoculum: Ingredients contaminated by a high level of micro-organisms cause
appreciable microbial degradation. However, inoculum size alone is not always a reliable
indicator of likely spoilage potential.
2. Nutritional factors: Gross microbial spoilage of a pharmaceutical product generally
requires appreciable growth in the dosage forms. The use of crude vegetable or animal
products in formulations provides an additionally nutritious environment.
3. Moisture content: Dissolved components of an aqueous formulation may form a
complex with water molecules. The high concentration of the solute in the formulation
indicates the water activity(Aw) is low. Condensed moisture films can form on the surface of
tablets or bulk oils.
4. Temperature: Spoilage of pharmaceuticals may occur over the range of about 10-
60C. Storage with specific temperature ranges the spoilage. Water for injections should be
at 80C prior sterilization, to prevent pyrogen production
5. pH: Extremes of pH prevent microbial attack, although growth of mould is commonly
observed in solutions of dilute hydrochloride acid. Preserved flavoured pharmaceutical
products can support growth of microbes.

9. 6. Redox potential: The ability of microbes to grow in environment is influenced by its
redox balance. Oxygen removal control spoilage in medicines. Due to high solubility of
oxygen emulsions become viscous.
7. Protective components: Various components of formulations and products may increase
the resistance and longevity of contaminant micro-organisms. Some substances such as
surfactants, suspending agents and proteins can increase the resistance of micro-organisms
to preservatives.

10. SOURCES AND TYPES OF MICROBIAL
CONTAMINATION
1. Atmosphere
2. Water
3. Raw material
4. Process operators
5. Equipment
6. Packaging
7. Buildings
The microbiological contamination of pharmaceutical products is influenced by the
environment in which they are manufactured and by the materials used in their formulation.

11. 1. Atmosphere: The number of micro-organisms in the atmosphere depends on the
activity in the environment and the amount of dust which is present. Air is not a natural
environment for the amount of dust is present. Micro-organisms are carried into the
atmosphere suspended on particles of dust, skin, clothing, droplets of moisture, sputum
from coughing & sneezing. Micro-organisms commonly from the atmosphere are bacteria
and fungi. E.g. Bacillus spp., streptococcus spp., Mucor spp., Penicillium spp. Etc.
2. Water: Water is one of the main constituents of many products and it is used for
washing and cooling process. Bacillus and Staphylococcus spp. may contaminate
softened water at the time of its preparation by base exchange method. Different types of
micro-organisms are present in fresh water and some microbes may contaminate the
water for sewage. E.g. E.coli, clostridium spp.
3. Raw materials: This are potentially rich source of micro-organisms. Synthetic raw
materials are usually free from microbes but contamination may occur. Raw materials
mainly contain bacteria and fungi e.g. Bacillus spp., salmonella spp., etc. products from
animal sources such as gelatine, desiccated thyroid, pancreas, etc. may be contaminated
with different pathogens.
4. Process operators: Micro-organisms may be transferred to pharmaceutical formulations
from process operators. Natural skin flora micro-organisms are the main source of
contamination. E.g. Sarcinna spp., Staphylococcus aureus, etc. other than this they may be
transferred from operators as a result of poor personal hygiene

12. 5. Equipment's: It should be simple as possible for the purposes required with minimum
of junctions, valves and pumps to allow cleaning in place by circulation of detergents or
other chemical antimicrobial agents. The type and growth depend on source of the
contamination, environmental conditions and nutrients.
6. Packaging: The microbial flora of packaging materials is dependent upon its
composition and storage conditions. Packaging material have a dual role and act both
to contain the product and to prevent the entry of micro-organisms or moisture. Containers
caps and closures may also be sterilized by moist heat, chemicals, gaseous agents or
radiation techniques
7. Buildings: Different molds and a few bacterial species are the most common flora of
ceilings and wall. To reduce microbial growth, all walls, floors and ceilings should be
smooth and washable. The risk of contamination and spoilage of a pharmaceutical product
during formulation, storage and administration to the patient is generally significant and
appreciation of this should be reflected in the formulation and design of the product.

13. ASSESSMENT OF MICROBIAL CONTAMINATION AND
SPOILAGE
Physical and chemical changes
Sterility test
Assessment of viable micro-organisms in non-sterile
products
Estimation of pyrogens
1.
2.
3.
4.

14. 1. Physical and chemical changes: Physical and chemical changes of different
pharmaceutical formulations indicate microbial contamination and spoilage. Change in
viscosity, pH, emulsion stability and loss of surface activity of formulations indicate
microbial spoilage. Measurement of oxygen consumption of the product can indicate the
degree of oxidative attack and microbial growth.
2. Sterility test: it is important that materials which are to be tested for sterility are not
subject to contamination from the operator during the course of test for this it should be
conducted by experienced personnel in an adequately clean room with laminar flow
cabinet facilities
3. Assessment of viable micro-organisms in non-sterile products: Non-sterile products are
tested for viable micro-organisms for detection of pathogens and total viable counts.
4. Estimation of pyrogen: A pyrogen is a material which causes rise in body temperature
when injected into a patient. The lipopolysaccharides and lipoproteins which comprise a
major part of cell wall of gram-negative bacteria. Two main procedures are used for the
detection of pyrogens.

15. THANK-YOU